RESUMO
The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells.
Assuntos
Apoptose , Mitocôndrias/enzimologia , Membranas Mitocondriais/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Humanos , Microscopia de Fluorescência , Mitocôndrias/fisiologia , Membranas Mitocondriais/fisiologia , PermeabilidadeRESUMO
The role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not well understood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubules were composed of alternating stretches of detyr- and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levels of detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for the morphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular to the apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.